DNA methylation helps regulate transcriptional activity and is widely studied in cancer biology. This investigation aimed to establish the significance of Human Papillomavirus (HPV) DNA methylation in HPV-associated disease both in terms of basic biology and as a potential biomarker.\ud Assays to assess DNA methylation and gene expression were developed and evaluated. Pyrosequencing was used to assess DNA methylation of four regions of the HPV16 genome (E2, L1/L2, enhancer, promoter). Gene expression was assessed using quantitative PCR with assays for E2, E6 and E7. HPV integration was assessed using Detection of Integrated Papillomavirus Sequences (DIPS).\ud The relationship between HPV methylation, gene expression and integration was explored in vitro and in vivo using cell cultures and clinical cohorts. A variety of sample materials were used including short term and immortal cell lines, cervical cancer biopsies, cytology samples and Vulval Intraepithelial Neoplasia (VIN) biopsies.\ud In general, hypermethylation of the HPV genome was associated with low HPV gene expression and the presence of integrated HPV genomes.\ud To better understand the potential clinical utility of HPV DNA methylation, the relationship between HPV DNA methylation and various stages of cervical disease was determined. The HPV genome was progressively hypermethylated with increasing severity of cervical disease and certain regions of the HPV genome were more affected than others. A longitudinal study was also performed in order to determine a relationship between HPV methylation and clinical outcome. Differences in HPV methylation among patients who had persistent HPV infection and low grade disease, persistent infection and high grade disease and patients that cleared HPV infections were observed. Throughout the study the potential application of a HPV biomarker was considered and the correct biomarker design procedures were referred to. Several of the early biomarker development steps were successfully achieved.

The NSC Study - DNA Methylation and Disease Grade .......................................96 5.1.2. Hypothesis 2: There is a Difference in DNA Methylation among the Different Disease Grades....................................................................................................................97 6.1.1. Hypothesis 1: DNA Methylation Varies among Regions of the HPV Genome and among the CpGs within Each Region................................................................................118 6.1.2.

Hypothesis 2: DNA Methylation of the HPV16 Genome Varied over Time.......119 6.1.3. Hypothesis 3: There are DNA Methylation Differences among the Outcome Groups ...........................................................................................................................127 6.1.4. Hypothesis 4: The Level of DNA Methylation of the CRISP Samples Should be Comparable to the Disease Grade Study..........................................................................134 6.1.5. Hypothesis 5a: There is an Association between DNA Methylation and Outcome Cytology ...........................................................................................................................135 6.1.6. Hypothesis 5b: There is a Relationship between Outcome Cytology and Outcome Histology ...........................................................................................................137 6.2.

1.1.3. HPV Genome and Gene Products 1.1.3.1. The Long Control Region

The LCR of HPV varies between types, however most HPV LCRs contain binding sites for the same subset of factors, the most significant of which appear to be YY1, CDP, Ap1, Sp1 and TBP (Spink and Laimins, 2005, O'Connor et al., 2000, Ai et al., 2000). For the promoter region, YY1 and CDP binding is associated with repression of P97. The expression of YY1 and CDP decreases upon differentiation alleviating these repressive effects (O'Connor et al., 1996, O'Connor et al., STUNKEL, W. & BERNARD, H. U. 1999. The chromatin structure of the long control region of human papillomavirus type 16 represses viral oncoprotein expression. J Virol, 73, 1918-30.